Cell separation apparatus and methods of use

ABSTRACT

The present invention provides automated devices for use in supporting various cell therapies and tissue engineering methods. The present invention provides an automated cell separation apparatus capable of separating cells from a tissue sample for use in cell therapies and/or tissue engineering. The cell separation apparatus can be used in combination with complementary devices such as cell collection device and/or a sodding apparatus to support various therapies. The automated apparatus includes media and tissue dissociating chemical reservoirs, filters, a cell separator and a perfusion flow loop through a graft chamber which supports a graft substrate or other endovascular device. The present invention further provides methods for using the tissue grafts and cell samples prepared by the devices described herein in a multitude of therapies including revascularization, regeneration and reconstruction of tissues and organs, as well as treatment and prevention of diseases.

RELATED APPLICATION INFORMATION

This application claims priority and is a divisional of U.S. Pat. No.9,144,583, filed Apr. 23, 2007, entitled CELL SEPARATION APPARATUS ANDMETHODS OF USE, that issued Sep. 29, 2015 and which is acontinuation-in-part of U.S. Pat. No. 8,202,725 filed Dec. 22, 2005,entitled CELL SODDING METHOD AND APPARATUS, that issued Jun. 19, 2012and which claims priority to U.S. Provisional Application No. 60/697,954filed Jul. 12, 2005, entitled AUTOMATED CELL SODDING METHOD ANDAPPARATUS, and U.S. Provisional Application No. 60/638,199 filed Dec.23, 2004 entitled CELL SODDING METHOD AND APPARATUS, each of which areincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is related to devices and methods for use insupporting various therapeutic procedures including cell therapies andtissue engineering.

BACKGROUND OF THE INVENTION

Cell therapy and tissue engineering is developing toward clinicalapplications for the repair and restoration of damaged or diseasedtissues and organs. In particular, the development of vascular grafts isa major goal in the field of cardiac and peripheral vascular surgery.Cardiovascular disease is the leading cause of mortality and morbidityin the first world. The standard of care, the autograft, is not withoutserious morbidity. Patients with systemic disease, leaving noappropriate autograft material or having already undergone auto grafts,numbering 100,000 a year in the United States alone, have few autograftoptions.

Researchers have thus been studying synthetic grafts for over 30 years.A major challenge is providing graft materials that are biocompatible.i.e., nonthrombogenic, non immunogenic, mechanically resistant, and haveacceptable wound healing and physiological responses (e.g.,vasoconstriction/relaxation responses, solute transportation ability,etc.). Furthermore, tissue graft materials should be easy to handle,store and ship, and be commercially feasible.

Vessels have two principal failure modes: mechanical and biological,caused by thrombosis within the vessel and subsequent occlusion and/orcellular ingrowth. Synthetic vessels having material properties capableof withstanding arterial pressure are commonplace, making the search fornon-thrombogenic materials the prime research interest. Endothelialcells obtained from the patient have been shown to decrease thethrombogenicity of implanted vessels(Williams et al., 1994, J. VascoSurg., 19:594-604; Arts et al., 2001 Lab Invest 81: 1461-1465).

Endothelial cells are of critical importance in establishing anon-thrombogenic cell lining within synthetic grafts. Thus, it isdesirable to achieve rapid cellular adhesion in or on a permeablematrix, scaffold, or other permeable cell substrate material in a matterof minutes or hours with an instrument that lends itself to theoperating room environment, maintains a sterile barrier, is easy to use,and produces consistent graft results.

Currently, there are four main approaches for meeting theserequirements, but with limited success: (i) the use of decellularizedtissue materials; (ii) the use of a self-assembly mechanism, whereincells are cultured on tissue culture plastic in a medium that inducesextracellular matrix (ECM) synthesis; (iii) the use of syntheticbiodegradable polymers, onto which cells are subsequently seeded andcultured in a simulated physiological environment; and (iv) the use ofbiopolymers, such as a reconstituted type I collagen gel, which isformed and compacted with tissue cells by the application of mechanicalforces to simulate a physiological environment (see, e.g., Robert T.Tranquillo, 2002, Ann. N.Y. Acad. Sci., 961 :251-254).

Pressure gradients involving transient high pressures have been used todeposit cells onto a permeable scaffold by a sieving action, i.e.,providing a bulk flow and using a substrate or scaffold material havingpores smaller than the cell population, thus capturing cells in thematrix (e.g., U.S. Pat. No. 5,628,781; Williams et al., 1992, J BiomedMat Res 26:103-117; Williams et al., 1992, J Biomed Mat Res28:203-212.). These captured cells have been shown to subsequentlyadhere to the scaffold material, but with only limited clinicalapplicability due to failure to fully meet the requisites for successfulgrafts discussed above, i.e., biocompatibility, mechanical strength, andnecessary physiological properties.

Beginning in the late 1970s, endothelial cell seeding was employedexperimentally to improve the patency of small diameter, polymericvascular grafts to counteract adverse reactions. Since that time,advances have been made toward this goal, with the majority of the focuson engineering a biological or a bio-hybrid graft.

Endothelial cells are more complex than was originally believed in thatthey do not merely create a single cell lining on the lumenal surface ofblood vessels. Endothelial cells also release molecules that modulatecoagulation, platelet aggregation, leukocyte adhesion, and vasculartone. In the absence of these cells, e.g., in the case of the lumen ofan implanted synthetic polymeric vascular graft, the host reactionprogresses to eventual failure. Loss of patency within the first thirtydays post-implantation is due to acute thrombosis. This early stagefailure is a consequence of the inherent thrombogenicity of thebiomaterial's blood-contacting surface, which is non-endothelialized. Todate, the only known completely non-thrombogenic material is anendothelium; any other material that comes into contact with thebloodstream is predisposed to platelet deposition and subsequentthrombosis. The long-term failure mode of small diameter polymericvascular grafts is anastomotic hyperplasia leading to a loss of patency.The precise mechanisms behind initiation of anastomotic hyperplasia arestill being defined; however, endothelial cell and smooth muscle celldysfunctions and improper communications are likely involved.

Early workers in the field of small diameter graft development sought topromote graft endothelialization and, thereby, increase patency bytransplanting a varying degree of autologous endothelial cells ontovascular grafts prior to implantation. This process has become known asendothelial cell seeding (partial coverage relying on continued cellproliferation) or cell sodding (full coverage). “Seeding” refers to aprocess which includes preclotting prosthetic surfaces with endothelialcells in platelet rich plasma (PRP). Sodding, by comparison, refers to aprocess which includes plating endothelial cells onto a pre-establishedPRP clot. Sodded graft surfaces are typically prepared utilizing atwo-step procedure. First, PRP is clotted onto a graft, incubated for aneffective period of time and then washed with culture media. Second, thePRP coated graft is plated with endothelial cells. In contrast, seededgraft surfaces are typically prepared using a one-step platingprocedure, whereby endothelial cells suspended directly in PRP areplated onto a graft surface. Accordingly, in a sodded graft, endothelialcells are plated onto the surface of a PRP clot, whereas endothelialcells are plated within the PRP clot in a seeded graft. Rupnick, et a!.,1989, J Vascular Surgery 9(6):788-795.

The underlying hypothesis is fairly simple; that is, by promoting theestablishment of the patient's own endothelial cells on the bloodcontacting surface of a vascular prosthesis, a “normal” endothelial celllining and associated basement membrane, together known as theneointima, will form on the graft and counteract the rheologic,physiologic, and biomaterial forces working synergistically to promotegraft failure. After 30 years of research in this area, includingpromising animal data, this simp Ie hypothesis has not yet yielded aclinical device.

The failure modes with endothelial-seeded grafts have been identical tountreated polymeric grafts, namely thrombosis and intimal hyperplasia.The failure modes, at least partially, are linked to the lack of afunctional endothelial layer, neo-intima, on the luminal surface of thegraft and/or abnormal endothelial and smooth muscle cell direct andindirect communication. These failures in early human trials camedespite successful demonstrations of seeded grafts developing into acell lining development. These data show that neo-intimal formation onpolymeric vascular graft lumenal surfaces in animal models occurs byendothelial cell proliferation from perianastomotic arteries, themicrovessels of graft interstices, or circulating progenitor endothelialcells not strictly from the seeded cells.

A potential source for endothelial cell seeding is microvascularendothelial cells (MVEC). Williams et al. pioneered both freshlyisolated and cultured human, canine, rabbit, rat, bovine and pigendothelial cells, specifically MVEC, in their laboratory to studycellular function. The source for human MVEC was aspirated tissue fromcosmetic liposuction. Two separate protocols for human fat MVECisolation were used depending on the end use of the cell population. Theprotocols differed in isolation complexity from a simple, operatingroom- compatible procedure for immediate sodding of human or animalgrafts to a more elaborate procedure if the MVEC will be subsequentlycultured.

The isolation of human MVEC has been enhanced by the use of liposuctionto obtain samples of human fat. The process of aspirating fat through aliposuction cannula dissociates subcutaneous fat into small pieces whichboosts the efficacy of the digestion process. The fat may be digestedwith collagenase (4 mg/cc) for 20 minutes, at 37° C. which releases >10⁶cells per gram of fat. These MVEC can be separated from the fat bygradient centrifugation. The MVEC will form a pellet and cansubsequently be resuspended in culture medium after discarding thesupernatant. These cells have undergone routine characterization todetermine the cellular makeup of the primary isolates. A majority of thecells isolated via this procedure are endothelial cells due to theirexpression of von Willebrand antigen, lack of expression of mesothelialcell specific cytokeratins, synthesis of angiotensin converting enzyme,prostacyclin and prostaglandin E2, synthesis of basement membranecollagens and the morphologic expression of micropinocytic vesicles.

A human clinical trial was undertaken to evaluate endothelial celltransplantation in patients requiring peripheral bypass. During thetrial, large quantities of endothelial cells were placed directly on thelumenal surface of an ePTFE graft. To improve cell deposition, allgrafts were pre-wetted in culture medium containing autologous serum.Cells were suspended in the same medium at a density of 2×10⁵ cells/cm²graft lumenal area. This solution was held at a cross-wall, ortransmural, pressure gradient of 5 psi to force cells onto the surface,a process termed “pressure sodding”. After institutional approval, 11patients were enrolled and received the experimental graft. Duringsurgical prep, the patients underwent liposuction to removeapproximately 50 grams of abdominal wall fat. The fat was processedusing the aforementioned procedure and the resulting cell population waspressure sodded on the intended graft and immediately implanted. Aftermore than 4 years of follow-up, these grafts have maintained a patencyrate similar to that of saphenous vein grafts.

Pressure gradients involving transient (<1 min.) relatively highpressures (250 mmHg) have previously been used to deposit cells onto apermeable scaffold by a sieving action, i.e., providing a bulk flow andusing a substrate or scaffold material having pores smaller than thecell population, thus capturing cells in the matrix (e.g., U.S. Pat. No.5,628,781; Williams et al., 1992, J Biomed Mat Res 26:103 117; Williamset al., J Biomed Mat Res 28:203-212.) However, despite theaforementioned advances, clinical coronary applicability has beenlimited to date because the vessels do not maintain sufficientlycohesive non-thrombogenic surfaces; research has focused on additionalmaturation time in vitro.

Endothelial cells are of critical importance in establishing anon-thrombogenic cell lining. In addition, a need still exists for anefficient and reliable method for producing endothelial cell linings ona synthetic graft in an operating room setting, and the currentinvention provides a solution. It is desirable to achieve rapid celladhesion in or on a permeable matrix, scaffold or other permeable cellsubstrate material in a matter of minutes or hours with an instrumentthat lends itself to the operating room environment, maintains a sterilebarrier, is easy to use, produces consistent graft results, and isinexpensive. The present invention enables the isolation of largequantities of endothelial cells from fat tissue and the rapid cellsodding of synthetic grafts, and enables automation and adhesion ofcells in a tum-key, operating room ready instrument for the rapidsodding of the graft. This invention will likely have other applicationsin addition to the lining of grafts for implantation.

SUMMARY OF THE INVENTION

The present invention provides devices for use in supporting variouscell therapies and tissue engineering methods. Specifically, the presentinvention provides a cell separation apparatus capable of rinsing andseparating cells from a tissue sample for use in cell therapies and/ortissue engineering. In a particular embodiment of the invention, thecell separation apparatus can be used in combination with a soddingapparatus to support autologous endothelialization of vascular graftsand endovascular devices.

In one embodiment, the cell separation apparatus comprising a mediareservoir; a cell processing device comprising at least one inlet and atleast one outlet, a first lobe and a second lobe, at least one pump, andat least one valve adapted to divert or prevent fluid flow, all of whichare in fluid communication with one another. In a preferred embodiment,the cell processing device comprises a centrifuge. In another embodimentthe cell processing device is disposable. In additional embodiments, thecell processing device further comprises an extraction tube and/or arotating coupling. In a particular embodiment, the rotating couplingfurther comprises a pressurized spray nozzle.

In another embodiment, the cell separation system of the presentinvention is designed to be modular such that components may be re-usedin other systems developed by the inventors. In an embodiment, the cellseparation device is adapted for use with a cell sodding device and/or acell harvesting device. In an embodiment, the apparatus is fullyautomated and may comprise, for example, a human machine interface, anelectronic graphical display, sensors, alarms, a cell counting device,and bar code reading device. In additional embodiments, the apparatusmay comprise a heater, a waste reservoir, or a tissue dissociatingchemical reservoir. In a specific embodiment, the cell separationapparatus is a handheld device.

In other embodiments, the apparatus may include one or more filters, forexample, between the cell processing device inlet and the tissuedissociating chemical reservoir; or between an outlet of the cellprocessing device and a sterile cell collection device. In oneembodiment, the filter excludes particles greater than about 100microns, and in another embodiment, the filter excludes particlesgreater than 30 microns. In a specific embodiment, the sterile cellcollection device is a syringe.

The media used in the present invention may be M199, M199E, PBS, Saline,and Di- Cation Free DPBS. In a preferred embodiment, the media is M199E.In another embodiment the tissue dissociating chemical is collagenase.

A kit is also provided for use in a cell therapy comprising the cellseparation apparatus of the present invention adapted for use with acell sodding apparatus, wherein the cell separation apparatus and cellsodding apparatus are contained within a durable enclosure. In oneembodiment, the kit comprises a flow path cartridge comprising one ormore fluid reservoirs, at least one inlet and at least one outlet; acell processing cartridge having at least one inlet and at least oneoutlet; an optional graft chamber cartridge for holding a graftsubstrate, the graft chamber cartridge having at least one inlet and atleast one outlet; at least one pump configured to cause flow through aflow path; at least one valve configured to direct flow from the cellseparator cartridge to the graft chamber cartridge; where the flow pathcartridge, cell separator cartridge and graft chamber cartridgecommunicate to form a continuous flow path, and wherein said flow pathcartridge, cell separator cartridge, and optional graft chambercartridge communicate with a modular kit enclosure capable of providingpower to the apparatus.

In one embodiment, the flow path cartridge, cell processing cartridgeand graft chamber cartridge are disposable. In another embodiment, thecell processing cartridge comprises a centrifuge. The apparatus of theclaimed invention can also be adapted for use with a cell maceratorwhich is in communication with the flow path cartridge.

In another embodiment of the present invention, the kit enclosurecomprises at least one sensor means for detecting the presence of theflow path cartridge, the cell processing cartridge and the graftchamber, and or at least one sensor means for monitoring and controllingtemperature, pressure and flow rate, wherein the sensor means is incommunication with an alarm.

Methods for preparing a tissue graft using the apparatus of the presentinvention are also provided in which media containing adherent cells isintroduced into the graft chamber, and a sustained low pressuretransmural flow of the media across the substrate for a time periodsufficient to adhere the cells to the substrate is applied. In aparticular embodiment the adherent cells are microvascular endothelialcells derived from adipose tissue. In another embodiment the endothelialcells are harvested from a patient to be treated with the apparatus ofthe present invention.

Additionally, methods for regenerating a tissue or organ in a subject byinjecting into the tissue or organ a cell suspension prepared by theapparatus of the invention are also provided. Methods for treating awound and preventing adhesion formation in a tissue or organ of asubject in need thereof by injecting into the tissue or organ at leastone cell suspension prepared by the apparatus of the invention are alsoprovided.

The present invention also provides an automated, sterile and safemethod and devices to form cells on a suitable graft for clinical use ina short period of time, as well as methods and devices for collecting asample of cells suitable for therapeutic use. The present inventionfurther provides methods for using the tissue grafts and cell samplesprepared by the devices described herein in a multitude of therapiesincluding revascularization, regeneration and reconstruction of tissuesand organs as well as treatment and prevention of diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. I is a schematic illustrating the system flow path of an embodimentof the cell separation apparatus.

FIG. 2 (A) depicts a cross section of the processing device of the cellseparation apparatus including the spray nozzle member and inner andouter centrifuge bowls in accordance with one embodiment of the presentinvention; FIG. 2 (B) provides a perspective view of the cell processingapparatus in accordance with one embodiment of the present invention;and FIG. 2 (C)-FIG 2 (F) depicts the twist locking mechanism which joinsthe processing device to the cell separation apparatus in accordancewith one embodiment of the present invention.

FIG. 3 (A) provides a perspective views in accordance with oneembodiment of the cell separation apparatus of the present inventionincluding the human-machine interface, the cell processing device(centrifuge), tube cassette, media bags, syringe pumps, pinch valves,collection syringe and barcode scanner; FIG. 3 (B) provides aperspective view of the cell separation apparatus of accordance withanother embodiment of the present invention; and FIG. 3 (C) provides aview of the rear of the cell separation apparatus in accordance with oneembodiment of the present invention.

FIG. 4 is a schematic illustrating the Clinical (OR) Kit inputs inaccordance with one embodiment of the present invention.

FIG. 5 is a schematic depicting the flow path of the Clinical (OR) Kitin accordance with one embodiment of the present invention.

FIG. 6 depicts the graft sodding module in accordance with oneembodiment of the present invention.

FIG. 7 depicts the cell collection module in accordance with oneembodiment of the present invention.

FIG. 8 is a perspective view of the cell collection module durablesconnected to the cell separation module with disposable componentsloaded for use in accordance with one embodiment of the presentinvention.

FIG. 9 (A) depicts a jet spray nozzle and rotating coupling inaccordance with one embodiment of the present invention; and FIG. 9 (B)depicts a spray nozzle member aligned with one lobe of the centrifugebowl in accordance with one embodiment of the present invention.

FIG. 10 (A) provides a perspective view of a pinch valve manifold rackin accordance with one embodiment of the present invention; FIG. 10 (B)provides a perspective view of the tubing rack of the pinch valvemanifold rack in accordance with one embodiment of the presentinvention; FIG. 10 (C) provides a cross sectional view of the tubingrack of the pinch valve manifold rack in accordance with one embodimentof the present invention; and FIG. 10 (D) provides a side view of thepinch valve manifold rack in accordance with one embodiment of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention are described herein in the contextof devices for use in supporting various cell therapies and tissueengineering methods. Those of ordinary skill in the art will realizethat the following detailed description of the present invention isillustrative only and is not intended to be in any way limiting. Otherembodiments of the present invention will readily suggest themselves tosuch skilled persons having the benefit of this disclosure. Referencewill now be made in detail to implementations of the present inventionas illustrated in the accompanying drawings. The same referenceindicators will be used throughout the drawings and the followingdetailed description to refer to the same or like parts.

In the interest of clarity, not all of the routine features of theimplementations described herein are shown and described. It will, ofcourse, be appreciated that in the development of any such actualimplementation, numerous implementation-specific decisions must be madein order to achieve the developer's specific goals, such as compliancewith application-related constraints, and that these specific goals willvary from one implementation to another and from one developer toanother. Moreover, it will be appreciated that such a development effortmight be complex and time-consuming, but would nevertheless be a routineundertaking of engineering for those of ordinary skill in the art havingthe benefit of this disclosure.

In accordance with the present disclosure, the components and processsteps described herein may be implemented using various types ofoperating systems, computing platforms, computer programs, and/orgeneral purpose machines. In addition, those of ordinary skill in theart will recognize that devices of a less general purpose nature, suchas hardwired devices, field programmable gate arrays (FPGAs),application specific integrated circuits (ASICs), or the like, may alsobe used without departing from the scope and spirit of the inventiveconcepts disclosed herein.

The present invention provides devices for use in supporting variouscell therapies and tissue engineering methods. Cell therapy, cellulartherapy, or cell-based therapy refers to the use of human or animalcells to replace or repair diseased or damaged tissue and/or cells, orto treat or prevent a disease or disorder.

Specifically, the present invention provides a cell separation apparatuscapable of digesting, rinsing, and separating cells from a tissue samplefor use in cell therapies and/or tissue engineering. As used herein,“cell rinsing” refers to the process of using additional fluid toresuspend cells that have been isolated from the fat/collagenasemixture. The resuspended cells can then undergo a second isolationprocess via centrifugation to purity the cell product (MVECs). Thisrinsing process reduces the concentration of digestion byproducts suchas, e.g., red blood cells, collagenase and proteins.

In a particular embodiment of the invention, the cell separationapparatus can be used in combination with a sodding apparatus to supportautologous endothelialization of vascular grafts.

Cell Separation Apparatus

In one embodiment of the present invention, the cell separation moduleor cell separation apparatus is a stand-alone piece of equipment thatcontains all necessary electronics and components to cut, heat, digest,and separate adipose tissue. In a preferred embodiment, the cellseparation component comprises a centrifuge. An outlet from the cellseparation module supplies a single cell suspension of isolated cells,to be connected to either the graft sodding module, cell collectionmodule, or other module.

FIG. 1 illustrates a system 100 and a fluid path (flow path) schematicfor a method of using a cell processing apparatus, including theinteraction between the centrifuge bowl of the cell processing apparatusand the fluid path. Referring to FIG. 1 by way of non-limiting example,adipose tissue (Fat) is manually pushed from a syringe 102 into acentrifuge bowl 104. The bowl 104 is then manually loaded into the CellProcessing Device onto the drive mechanism which utilizes a twist lockcoupling to secure it. A Centrifuge Chamber lid is then closed and hotair circulating within this chamber keeps it warm to, in one example,about 37° C. The system 100 may further include a plurality of valvesV1-V10, a first syringe S2, a first vent 108, and a second vent 110. Thesystem 100 further comprises consumables including a waste bag 106, asecond syringe S2, in addition to a fluid tubing matrix (not shown),bags of fluid 112 (e.g., PBS or Serum). In an embodiment, the valvesV1-V10 are pinch valves and block flow by pinching the tubing within thetubing matrix. Valves V1-V3 may be disposed in series, valves V1 and V2maybe be coupled to the centrifuge bowl 104 and in communication with V3which is coupled to the vent 110 and the second syringe S2. Valve V4 maybe in communication with valve V5 and the second syringe S2, the serum112 and/or media 114 may be connected to valves as appropriate forprocessing Valve V9 may be in communication with the waste container 106as well as valves V8 and V10, valve V10 may be in communication as wellwith the first syringe S1 and the vent 108.

In an embodiment, a 60 ml syringe filled with chilled (to about 2° C.)collagenase solution is loaded into the syringe driver S1. A heatingelement integral to the syringe driver S1 but not shown warms thissolution to about 37° C., within approximately 15 minutes. After thecollagenase solution has been heated to about 37° C., the syringe driverS1 is activated and the collagenase solution is pushed through valve V7into the Centrifuge Bowl 104 in which the adipose tissue from 102 waspreviously disposed. In an embodiment, a filter F may be disposedbetween valves V7 and V8 and, in this example, the valve V5 remainsclosed to block flow through the 100 μm filter (F). The centrifuge bowl104 drive mechanism (motor) oscillates the bowl 104 for an amount oftime sufficient to allow for the collagenase to “digest” the adiposetissue. In a preferred embodiment, the amount of time sufficient toallow for the collagenase to digest the adipose tissue is approximately30 minutes.

Fibrous tissue is then removed from the digested material when thesyringe S1 draws back 50 ml of fluid via the valve VS. The fibroustissue is collected by the filter (F) between valves V7 and V8. In oneembodiment, the filter F excludes material greater than 100 μm in size.The first syringe S1 pushes the first half of fluid temporarily to thewaste tank 106 (which is pristine for this step). A second pull, e.g., asecond pull by the syringe S1, is used to complete the evacuation of thecentrifuge bowl 104 and filter all material from it via the filter F.The valves V7 and V8 are then aligned to push the filtered material backinto the bowl via valve V7. In one embodiment, the second half of thefluid comes straight from the syringe pump S1 and the first half isdrawn from the waste bag 106 back into the S1 syringe and then pushedback into the centrifuge bowl 106.

In an embodiment, the centrifuge bowl 104 is then spun at about 3100 RPMfor about 5 minutes. During centrifugation, the endothelial cells areseparated from the digested material and deposited in the two lobes ofthe centrifuge bowl 104. The cells will tend to “pack” into the lobesand remain in the lobes until pushed out of them via separate means. Inthis example, and with the centrifuge bowl still spinning at about 3100RPM, additional MI99E fluid is pushed from the second syringe S2 intothe bowl 104 via valve V2. This M199E fluid 114 combined with thespinning motion displaces less dense fat cells to the center of the bowl104. At least one notch aperture is located near the top center of thecentrifuge bowl 104 through which fat is directed from the inner bowlinto an outer chamber via the centrifugal action of the inner bowl. Thiseffectively removes much of the spent fat tissue from the inner bowl.

In an embodiment, the centrifuge bowl 104 is brought to rest and thecollagenase/M199E mixture settles to the bottom of the inner bowl. Thisspent fluid is then sent to waste 106 using the syringe pump SI viavalves V7 and V9. In some embodiments, fresh media MI99E 114, forexample, from is added to the bowl 104 via valve V2. A light spin isperformed to “rinse” the bowl 104. The fluid resulting from this rinseis then sent to waste 106 using syringe pump SI via the valves V8 andV9.

In an embodiment, when the inner bowl is empty and rinsed, the cellpellets still located in the centrifuge lobes are pushed back into theinner bowl via a rotating (or rotary) coupling and fluid moved throughtubing. In an embodiment, the rotating coupling comprises at least onetransport tube for use in adding and/or removing liquid from the innerchamber of the centrifuge bowl. In one embodiment, the rotating couplingcomprises a transport tube for adding liquid to the inner chamber of thecentrifuge bowl, and another transport tube for removing liquid from theinner chamber of the centrifuge bowl.

The suspended cell product is removed via V6 into the third syringe SO,and the cell product is then sent to a sterile container (not shown)attached to V5. In an embodiment of the invention, the sterile containeris a syringe. In an additional embodiment, a second filter (not shown)will typically be used between valve V5 and the container to remove andresidual particles greater than about 30 microns.

Centrifuge bowl of the cell processing apparatus is a lobed, two chamberconstruction consisting of an inner bowl and an outer bowl as shown inFIGS. 2A and 9B. The outer bowl is used primarily as an overflow chamberto store spent fat during the cell separation process. The novel designof the Centrifuge Bowl of the present invention provides optimized dualfunctionality. For instance, the inner chamber of the bowl is configuredto provide a mixing zone, which is utilized in the digestion step of thecell processing methods disclosed herein, as well as a separation zone,(i.e., the lobes of the inner bowl) which optimizes the capture of asufficiently purified cell pellet.

The lobes of the inner bowl of the present invention are specificallyconfigured to optimize the collection of a endothelial cells andminimize collection of non-endothelial cell materials such as, forexample red blood cells and other cell fragments. The centrifuge bowlillustrated in FIGS. 2A-2F and 9B comprises a twist lock coupling memberwhich can be utilized to quickly and efficiently couple and uncouple thecentrifuge bowl to the cell separation device.

The centrifuge bowl of the cell processing apparatus and fluid path isshown in FIGS. 2A-2F. In particular, FIG. 2A depicts a cross section200A of the processing device of the cell separation apparatus includingthe spray nozzle member and inner 402 and outer 404 centrifuge bowls inaccordance with one embodiment of the present invention. FIG. 2Bprovides a perspective view 200B of the cell processing apparatus inaccordance with one embodiment of the present invention. FIGS. 2C-2Fillustrate the twist locking mechanism, FIG. 2C illustrates embodiment200C of the cell separation apparatus, including a bottom portion 208comprising a twist-locking mechanism 206, FIG. 2D 200D illustrates a topportion 210 and a locking mechanism 212 of the top portion 210 disposedin proximity to the locking mechanism 206 of the bottom portion 208 butnot coupled to or assembled with the bottom portion 208. FIG. 2Eillustrates the top portion 210 and a bottom portion 208 where thelocking mechanism 206 of the bottom portion 208 is engaged with the topportion via a plurality of channels 214 on the top portion 210 that arein communication with the locking mechanism 212. FIG. 2F illustrates thetop portion 210 engaged with the bottom portion 208 via the respectivelocking mechanisms 206 and 212 after the locking mechanism 206 entersthe window 214 and the top portion 210 is rotated to position 206 in thechannel of 212.

FIGS. 10A-10D depict a pinch valve manifold rack 1000 in accordance withone embodiment of the present invention. Preferably, the valves are partof the durable instrument and do not contact the fluid directly. In oneembodiment, the device is designed to hold and process about 60 ml ofadipose tissue and 60 ml of collagenase solution.

FIGS. 3A-3D illustrate various view of the cell separation apparatus300. FIGS. 3A-3C show the cell separation module durable and disposablecomponents including the human machine interface, cell processing device(centrifuge), tube cassette, media bags, syringe pumps pinch valves,collection syringe and barcode scanner. FIG. 3A provides a perspectiveviews in accordance with one embodiment of the cell separation apparatusof the present invention including the human-machine interface, the cellprocessing device (centrifuge), tube cassette, media bags, syringepumps, pinch valves, collection syringe and barcode scanner; 3B providesa perspective view of the cell separation apparatus of accordance withanother embodiment of the present invention; and 3C provides a view ofthe rear of the cell separation apparatus in accordance with oneembodiment of the present invention. In one embodiment, the cellseparation module durable unit houses all of the electronics necessaryfor operation of the device, including the computer boards, software,power supply, and an user interface. In a preferred embodiment, the userinterface includes an LCD screen with buttons that guides the userthrough the set-up and operation of the device. The cell separationmodule durable can also house the necessary pinch valves, motors,sensors and other durables required for cutting, heating, digesting, andcentrifuging the subject tissue. In a preferred embodiment, the subjecttissue is adipose tissue. Pinch valves protrude from the enclosure on atop flat surface to allow valves to engage the disposable fluid pathway.In a preferred embodiment, electronics are located a maximum distancefrom any fluid pathways.

In another embodiment, the device includes a mountable hook to hangmedia and waste bags. Preferably, the bag hook is mounted to either thegraft sodding durable or the cell collection durable to maximize thedistance between the media bags and electronics housed in the cellseparation durable. This separation reduces risk of electronics damagefrom fluid spills

In a particular embodiment of the invention, all elements of the cellseparation module flow path are disposable. In one embodiment, thesedisposable components can be assembled on a rigid tray that loads ontothe cell separation module durable. The user loads the disposable trayby placing the tray onto the flat surface of the durable by aligning thepinch valves with the valve cutouts in the disposable tray. The userthen slides the tray forward to engage tubing loops in the pinch valvesand lock the disposable tray in place. All disposable components arelocated in the tray to align with and engage the necessary durablecomponents in the cell separation durable by this loading operation. Thetray design minimizes the user's burden for set-up and disposal byeliminating the need for many tubing connections and individual loadingof many disposable components. After loading the tray, the user can loadthe disposable centrifuge bowl into a recess provided in the durablecomponent and attach inlet and outlet tubing from the disposable tray tothe centrifuge, media bag, waste bag, and sodding or collection unit.

In accordance with another particular embodiment of the invention, cellpellets located in the centrifuge lobes are flushed from the lobes usinga pressurized jet of fluid (jetspray) introduced from a nozzle member.In a particular embodiment, the nozzle member is in communication with arotating coupling specifically adapted for use with the nozzle. FIG. 9Aillustrates an embodiment 900A of the jet spray nozzle and the rotatingcoupling in accordance with a specific embodiment of the invention. FIG.9B further illustrates an embodiment 900B of the rotating coupling andjet spray nozzle aligned in the centrifuge bowl.

By way of non-limiting example, the jet spray nozzle discharges fluidwhich impinges on a cell pellet “packed” or lodged into the centrifugelobe. The cell pellet is broken up and/or dislodged from the lobe andfluid and cell pellet material are carried back to the bottom of thecentrifuge bowl via gravity. In one embodiment, the jet spray nozzle isaligned with a support structure in the cell processing apparatus to fixits location (see e.g., FIG. 9B). In a preferred embodiment, thecentrifuge motor is controlled by a computer and is adapted to indicatethe position of the centrifuge bowl. Thus, the motor is capable ofrotating the centrifuge bowl to align the jet spray nozzle with eachlobe of the centrifuge bowl. For instance, after one lobe flush, thecentrifuge bowl is rotated 180 degrees and the jet nozzle is activatedto flush the second lobe. Accordingly, the jet spray nozzle is capableof efficiently dislodging the cell pellet in each lobe of the centrifugebowl.

The fluid used for this purpose may be a fluid with a physiologicalconcentration of sodium chloride at a physiological pH. In a preferredembodiment, the fluid used for this purpose is a 6:1 ratio of MI99E andSerum, respectively. Serum is used to de-activate any residualcollagenase in the cell product. Approximately 1 ml of cell material and10 ml of M199E/Serum mixture is now in the bottom of the inner bowl.

In an embodiment of the present invention, the centrifuge lobes areadapted to include one or more selective filtering devices capable ofusing centrifugal force to concentrate or select out particular cellpopulations. In an embodiment, the selective filtering device(s) may beprovided preferential alignment with the jet spray nozzle to recover thedesired concentrate or cell population. In another embodiment, the cellseparation apparatus is adapted to include one or more selective filtersupstream of the collection module which are capable of selecting andcapturing the desired cells, rerouting any excess media, and allowingthe desired cells to be collected at a desired cells/ml concentration.In another embodiment, the cell separation apparatus of the presentinvention may include a cell counting and/or cell sorting deviceincluding, but not limited to, devices using known optical density ororifice electrical stimulation technology. Preferably, the device isdivided into the three distinct modules: a cell separation module, agraft sodding module, and a cell collection module.

The cells to be processed by the cell separation apparatus of thepresent invention may include, for example, fibroblasts, smooth musclecells, pericytes, macrophages, monocytes, plasma cells, mast cells,adipocytes, tissue-specific parenchymal cells, endothelial cells,urothelial cells, adipose derived stem cells and various other celltypes encountered in tissue engineering applications and cell therapies,including undifferentiated adult stem cells from various tissue sources.Mitchell, J B. et al., Immunophenotype of Human Adipose-Derived Cells:Temporal Changes in Stromal-Associated and Stem Cell-Associated Markers,Stem Cells 2006, 24:376-385; McIntosh K. et al., The Immunogenicity ofHuman Adipose-Derived Cells: Temporal Changes In Vitro, Stem Cells2006,24:1246-1253; Kern S. et al., Comparative Analysis of MesenchymalStem Cells from Bone Marrow, Umbilical Cord Blood, or Adipose Tissue,Stem Cells 2006,1294-1201. In a preferred embodiment, the cells areendothelial cells, more preferably human microvascular endothelial cellsobtained from autologous microvascular rich adipose tissue as referredto in U.S. Pat. No. 4,820,626 (by Williams et al., issued Apr. 11,1989), U.S. Pat. No. 5,230,693 (by Williams et al., issued Jul. 27,1993), and U.S. Pat. No. 5,628,781 (by Williams et al., issued May 13,1997), all of which are hereby incorporated by reference in theirentireties. The adherent cells may be autologous, allogeneic, orxenogeneic, but preferably are autologous in origin.

Graft Sodding Module

FIG. 6 illustrates a graft sodding module 600 including the durable anddisposable components. As used herein, the term “graft sodding module”refers to the durable and disposable components that are employed toapply the cells provided by the cell separation unit onto a porous graftscaffold using a pressure sodding technique. The cell separationapparatus of the present invention is designed to be modular such thatcomponents may be used and re-used with other devices and systems. Inone embodiment, the cell separation apparatus is adapted for use with acell sodding device or graft sodding module.

In an embodiment of the invention, the sodding module contains twodurable components: the sodding unit durable and the graft chamberdurable. These durable components physically mate with the cellseparation durable to provide a power and communication connection. Inanother embodiment of the invention, the sodding module durables arecontrolled by the electronics in the cell separation module durable. Thegraft chamber durable provides secure mounting for the disposable graftand houses components necessary for heating of the chamber. The soddingdurable contains the hardware (e.g., pinch valves, sensors) that isspecifically required to manipulate flow through the graft chamber asneeded for the pressure sodding application. In one embodiment, thesodding durable has a top flat surface with protruding durable equipmentwhere the sodding disposable can be loaded.

In a further embodiment, sodding disposable components include thedisposable graft chamber and a sodding disposable tray. The scaffold orother substrate material is typically preloaded in the disposable graftchamber, which provides a sealed environment for delivery of liquids tothe graft while prohibiting all other gaseous, liquid, and solid matterexchange with surroundings.

In an embodiment, the sodding disposable rigid tray includes alldisposable components and connecting materials required for the soddingoperation. The tray loads onto the flat surface of the sodding durableby aligning the pinch valves with the valve cutouts in the disposabletray and sliding forward to engage tubing in the pinch valves. The userconnects the cell separation disposable, sodding disposable, and graftchamber disposable to form the complete flow path for sodding.

The separation and sodding media may be a commercially available mediaincluding DMEM, F12, AlphaMEM, University of Wisconsin Solution, etc.,or any combination thereof, without or without additional factors, whichmay include heparin or other factors that accommodate the desired celltype.

Collection Module

FIG. 7 illustrates a cell collection module 700 durable 702 anddisposable 704 components. In an embodiment of the present invention,the cell separation apparatus is also designed to function with acollection module or collection device. The collection module orcollection device refers to the durable and disposable components thatare necessary to collect cells from the cell separation unit in asyringe for use in cell therapies.

FIG. 8 illustrates an embodiment 800 of durables connected to the cellseparation module with disposable components loaded for use. In anembodiment, the collection module durable physically mates with the cellseparation unit to provide a power and communication connection. Thecollection durable houses a linear actuator that interfaces with asyringe to automatically collect the cell product produced in the cellseparation unit.

In another embodiment, the disposable component in the collection unitis the syringe to collect the cell product. The syringe is held in placeby a clip on the collection unit durable. The top of the syringe isloaded into the durable such that the syringe plunger can be drawn bythe motion of the actuator. The user connects the outlet tube from thecell separation module to the tip of the syringe.

Overview of Clinical (OR) Kit System

The clinical kit or operating room (OR) Kit of the present inventionprovides a sterile flow path, through which adipose tissue can bedigested, separated, and pressure sodded onto a porous vascular graftscaffold. The system is also capable of pretreating the graft scaffoldto prepare it for the pressure sodding operation. In one embodiment, theflow path comprises three disposable cartridges that interlock with adurable Clinical (OR) Kit system enclosure. The disposable cartridgesinclude a flow path cartridge with fluid reservoirs, a disposablecentrifuge cartridge comprising the cell separation apparatus of thepresent invention, and a disposable graft chamber that is pre-loadedwith a graft scaffold. The Clinical (OR) Kit system is a self-contained,stand-alone system requiring only power to operate.

The system of the present invention is designed to require minimaloperator interaction. The sterile graft chamber with preloaded graftscaffold, flow path cartridge, and centrifuge cartridge can be loadedinto the Clinical (OR) Kit. The flow path cartridge can be preloadedwith media which may be, for example, M199, M199E, PBS, Saline, andDi-Cation Free DPBS. In a preferred embodiment, the media is M199E.

The operator can then inject reconstituted collagenase from the hospitalpharmacy, serum separated from the patient's blood and adipose tissuefrom the patient into the centrifuge and flow path cartridges throughthe appropriate injection ports. After completing this system set-up,the operator can start a sodding operation using an LCD interface on theClinical (OR) Kit. With no additional interaction from the operator, theClinical (OR) Kit will automatically perform all operations necessary toprepare a M199E/serum solution, pretreat the graft, digest adiposetissue using an externally prepared collagenase 1PBS solution,centrifuge to isolate target cells, pressure sod the target cells intothe porous graft scaffold, purge excess cells from the graft lumen,recirculate M199E/serum solution over the sodded graft, and isolate flowto the graft for harvest.

FIG. 4 illustrates the inputs into the Clinical (OR) Kit durableenclosure for the graft processing operation. Components of theenvisioned Clinical (OR) Kit system as illustrated in FIG. 4 include butare not necessarily limited to: an Clinical (OR) Kit enclosure 402, afront panel display (FPD), an Clinical (OR) Kit flow path cartridge 404,a graft chamber 406 with pre loaded scaffold, a main controller board(MCB) (not shown), an analog board (not shown), a centrifuge andcentrifuge cartridge 408, at least one pump (not shown), a fluiddistribution system (not shown), various sensors and alarms (not shown),and a cell counter (not shown). Also illustrated in FIG. 4 are theinputs comprising a patient's serum 410, a patient's adipose tissue 412,and collagenase reconstituted in PBS 414. As well as an output of thesystem 400 the endotheliazed graft scaffold that is ready to implant416.

The Clinical (OR) Kit enclosure 402 refers to the mechanical platformfor the Clinical (OR) Kit which supplies power and gives mechanicalstability to the Clinical (OR) Kit. The enclosure allows for entry cableconnection for power. In a particular embodiment, this enclosure alsohouses all durable components for the instrument including motors, pinchvalves, front panel display and sensors.

The Front Panel Display (FPD) provides a user-friendly graphical LCDdisplay. The screens on the FPD display allow the operator to performall the functions necessary to complete a pressure sodding operation inor adjacent to the OR. The operator will have the ability to begin agraft processing operation and view the status of the graft preparationat any time, but is restricted from changing parameters that mayinfluence the quality of the sodded graft.

The flow path cartridge 404 refers to the disposable, self-containedentity through which fluid flows throughout the system. The flow pathcartridge 404 includes a flow circuit, pump disposables, and fluidreservoirs for both feed and sump. The flow path cartridge 404 mateswith the cell processing or centrifuge cartridge 408 and a graft chamber406, which houses graft for pressure sodding in the operating room. Theflow path cartridge 404 physically mates with the enclosure 402. In analternative embodiment, the disposable cell processing cartridge may beincluded as part of the flow path cartridge 404. In another embodiment,the pump disposable is separate from the flow path cartridge 404.

FIG. 5 provides a conceptual illustration 500 of the pathway through theflow path cartridge 404. In another particular embodiment of the presentinvention, the device system is modular, such that the tissue digestionand separation portion of the device can be used with interchangeablemodules to either apply cells to a vascular graft or collect cells in asyringe. FIG. 5 shows the device assembled in a modular system. Becausethe cell separation portion of the device is housed in a distinct,separate unit, this embodiment also provides flexibility for pairing thecell separation unit other with other systems.

As shown in FIG. 5, the graft chamber 514 houses the graft scaffold forthe graft processing operation. The scaffold is preloaded in the graftchamber 514, which provides a sealed environment for delivery of liquidsto the graft while prohibiting all other gaseous, liquid, and solidmatter exchange with surroundings. FIG. 5 further illustrates theadipose tissue 502 and the collagenase reconstituted in PBS 504, both ofwhich may be disposed in the centrifuge disposable 506. The centrifuge506 may be in communication with a waste unit 508 via a port 522E and incommunication with an M199 supply 510 via a port 522A. The M199E supply510 may be mixed with serum 512, and the supply 510 is in communicationwith the recirculation line 516 via ports 522B and 522D. The centrifugedisposable 506 may be in fluid communication with the graft chamber 514via ports 522E and 522C. The graft chamber 514 may be in fluidcommunication with the transmural outlet 518 and the liquid outlet 520,which is also in fluid communication with port 522F and the graftchamber 514. The waste unit 508 may be in fluid communication with therecirculation line 516 and the liquid outlet via port 522F. In oneembodiment, three ports 522C, 518, and 520 on the graft chamber 514connect with tubing from the sodding disposable tray to provide inlet522C, transmural outlet 518, and lumenal outlet (liquid outlet) 520 fromthe graft chamber 514. In an embodiment, the graft chamber 514 restsinside the chamber durable which has a closing door to enclose thechamber during the sodding operation.

The graft substrate (“scaffold”) materials used in the present inventionmay be any preferably permeable material of various sizes andgeometries. The material may be natural or synthetic materials,including, but not limited to, polyethyleneterathalate, polyurethane, orexpanded poly-tetrafluoroethylene (ePTFE). In another embodiment, thegraft scaffold may be a biopolymer, such as collagen. The material maybe preclotted and/or elastin, or allograft vessels, such ascryopreserved vein, decellularized vein or artery. In yet anotherembodiment, the scaffold may be a composite material such as an elastinscaffold with a polymeric coating, for example electrospun on thesurface to improve mechanical properties. The material may be preclottedor pre-treated with a protein (e.g., albumin) or plasma, which incertain embodiments can serve to further enhance the adherence,spreading, and growth of tissue cells on the substrate material. Thegraft substrate or scaffolds may be constructed by any suitable method,including, but not limited to, those referred to in Liu, T. V. et al.,2004, Adv. Drug. Deliv. Rev. 56(11):1635-47; Nygren, P. A. et al., 2004,J. Immunol. Methods 290(1-2):3-28; Hutmacher, D. W. et al., 2004, TrendsBiotechnol. 22(7):354-62; Webb, A. R. et al., 2004, Expert Opin. Biol.Ther. 4(6):801-12; and Yang, C. et al., 2004, BioDrugs 18(2):103-19.

The main controller board (MCB) in the Clinical (OR) Kit includes amicroprocessor core module with appropriate interface to the analogboard, which controls the peripheral sensors in the Clinical (OR) Kitassembly. Software resides on the main controller board processor whichprovides a straight forward user interface that ensures reliable anddeterministic operation of the Clinical (OR) Kit. The analog board isperipheral to the MCB and is used to drive actuators and receive andcondition sensor information.

A centrifuge separates the cells before pressure sodding into the graft.In one embodiment, the wetted centrifuge bowl is a separate disposablecentrifuge cartridge, and the durable components are housed in theClinical (OR) Kit enclosure.

One or more pumps drive flow through the system which are designed tokeep pressure pulsations to a minimum. The wetted pump components arepart of the flow path cartridge, and the pump shaft is driven bynon-invasive means. In one embodiment of the present invention, thepumps are automatically self-priming during operation of the cellseparation device

The Clinical (OR) Kit system is designed to automatically advancethrough flow pathways necessary to pretreat a graft scaffold, prepareadipose tissue for sodding into the graft, apply the cells to the graft,and recirculate a M199E/serum solution over the graft to maintainviability until harvest. In one embodiment, tissue preparation includestreatment with collagenase that has. been reconstituted from thepowdered from outside of the Clinical (OR) Kit using a PBS solution,followed by centrifugation. The cells are then automatically resuspendedin MI99E/serum solution that is stored in a fluid reservoir within theClinical (OR) Kit before the solution is applied to the graft. Fluidvalves are configured to create these necessary pathways within the flowpath cartridge and centrifuge cartridge and are controlled by Clinical(OR) Kit software. A constant pressure across the graft scaffold ismaintained during the pressure sodding operation.

In a particular embodiment, the Clinical (OR) Kit includes sensorsnecessary to monitor and control temperature, pressure, and flowrate.Different flow path cartridges and graft chambers are loaded into theClinical (OR) Kit to match the specific type of graft sodding to becompleted (e.g. CABG, peripheral). The sensors are capable of detectingthe presence of the flow path cartridge, centrifuge cartridge, and graftchamber to ensure the disposables are properly loaded. Additionally, thesensors are capable of detecting the type of flow path cartridge andgraft chamber loaded to ensure the correct disposables are used.

The pressure sodding operation requires that 200,000 cells are appliedto the graft scaffold for each cm² of scaffold.

The Clinical (OR) Kit is capable of accepting input of collagenasereconstituted with PBS solution. In one embodiment, the kit accommodatesat least about 60 mL of prepared collagenase solution. The kit acceptsinput of adipose tissue. In an embodiment, the adipose inletaccommodates between about 30-60 mL of adipose tissue. In anotherembodiment, the adipose inlet is positioned to allow the tissue to beintroduced into an environment that is preheated to 37° C.

In an additional embodiment, the Clinical (OR) Kit system is capable ofcutting adipose tissue using a consumable cutting adapter that can beoptionally used depending on the tissue source. In a particularembodiment, the consumable cutting adapter is compatible for connectionto the Tulip syringe. The system is additionally capable of heating thegraft chamber, spaces where adipose tissue is loaded prior to digestion,and spaces for digestion to 37° C., and metering a volume of collagenasesolution equal to the expected adipose tissue input.

The system is capable of mixing an adipose tissue and collagenase cellslurry. In a preferred embodiment, the system carries out the mixing andseparation operations in a single centrifuge disposable, i.e. the cellprocessing device, that mates with the flow path cartridge and graftchamber. The system is also capable of removing fibrous material fromthe digested mixture. In an embodiment, the maximum allowable particlesize in the resuspended cells does not exceed about 100 mm.

In an embodiment, the Clinical (OR) Kit system is capable of isolating avolume of “target cells” from an adipose tissue that has been digestedby collagenase, and collecting the isolated target volume fromseparation. In a preferred embodiment, the system provides the followingtarget pellet volume purity: less than 5% by volume of total isolatedpellet volume for red blood cells; less than I % by volume of totalisolated pellet volume for adipose cells; less than 4% by volume oftotal isolated pellet volume for dead cells. In an additional preferredembodiment, all particles in the resuspension have a diameter less thanor equal to 100 mm. In yet another preferred embodiment, the separationprocess does not expose the cells to a force greater than 9000.

In an embodiment, the target cells are resuspended in a 6:1 volumetricmixture of MI99E and serum. In another embodiment, the system providesmeans to control the number of cells applied to the graft scaffold, witha target number of around 200,000 cells/ cm² graft. Variation in thistarget number of +50% to −10% is acceptable. By way of example, theClinical (OR) Kit system uses a volume of 6: I MI99E/serum solution thatis proportional to the expected volume of adipose tissue loaded into thesystem.

In an embodiment, the system includes a disposable graft chamber that ispreloaded with a graft scaffold for sodding. The graft chamber iscapable of accommodating graft scaffolds with lengths from about 1-90cm; graft inner diameters sizes from about 1-12 mm; and graft wallthickness from about 100-700 microns. The graft chamber provides asealed environment for sodding which prohibits gaseous, liquid, andsolid matter exchange with surroundings, except through graft chamberports.

All individual disposable components of the system are adapted to matewith each other to form a continuous flow path. Further, all disposablewetted materials and coatings of the system of the present invention arebiocompatible, and designed to withstand gamma irradiation to 25-40 kGywith at least 5% transmittance of clarity post-sterilization.Additionally, all non- disposable materials and coatings of the system,with the exception of internal electrical components, are compatiblewith typical disinfecting solutions including, for example, Cidex(glutaraldehyde antiseptic solution), 70% ethanol, 100% Isopropylalcohol, and 10% bleach solution.

In one embodiment, the Clinical (OR) Kit system includes an electronicsmodule with control electronics capable of driving, conditioning,acquiring and processing sensors for pressure, temperature, andflowrate. Pressure is measured at the graft chamber. In one embodiment,pressure across the scaffold wall is controlled at a target value offrom about 1.5 psi, not to exceed 2.0 psi. In another embodiment,temperature is measured and maintained at about 37° C. in spaces fordigestion and in the graft chamber. In another embodiment, flowratemeasurement and/or control is implemented as needed to maintain thepressure requirement across the wall of the graft.

In one embodiment of the invention, software is resident in a centralprocessor which controls electrical components and communication pathscontained within the device enclosure. The system of the presentinvention is adapted such that the software prevents operation of anyequipment if all disposable components are not correctly connected andinterlocked in the enclosure.\

Clinical (OR) Kit Operation

The Clinical (OR) Kit system automatically advances through flowpathways necessary to pretreat a graft scaffold, prepare adipose tissuefor sodding into the graft, apply the cells to the graft, purge thegraft lumen, and recirculate M199E/serum solution over the graft tomaintain viability until harvest. This flow path is illustrated in FIG.5.

Clinical (OR) Kit Operation Setup

The illustrative systems described herein will typically include amicroprocessor and associated software to control the system andautomate one or more steps based on user input. The software may allowfull or partial automation of, for example, controlling flow throughtubular conduits by controlling pumps and valves, controllingtemperature, and controlling cell separator and macerator devices.Preferably the system is fully automated, but capable of beingreconfigured based on one or more input parameters. The systems mayfurther include various sensors to detect or measure system parameters,such as pressures that would indicate a blockage, and signal same to themicroprocessor or user. In one embodiment, the system is a hand-heldsystem.

The controlled, sustained differential pressure gradient across thepermeable scaffold material may be created by any suitableconfiguration, including, but not limited to, gear pumps, peristalticpumps, diaphragm pumps, centrifugal pumps, and passive pressure headscreated by a column of fluid, so long as the pressure is sufficientlysustained and at a magnitude sufficient to achieve the advantages of theinvention. In a particularly preferred embodiment, the pressure isapplied transmurally to a vascular graft scaffold using media containingendothelial cells at a pressure head of about 50 mmHg and for a durationof about 5 minutes.

Because at least a portion of the flow for the current invention istypically transmural, the flow rate is dependent upon the permeabilityof the graft material, and decreases as the cells are applied to theluminal surface. Transmural flow rates before the introduction of cellscan be from 5-50 ml/min depending on the graft material and generallydecrease to 1-10 ml/min after the introduction of cells. Preferredendothelial cell numbers include 120,000-2,000,000 cells/cm² of luminalsurface area, more preferably about 250,000 cells/cm².

In one embodiment, the user installs the durable components required forthe current application (i.e. graft sodding durables or cell collectiondurables) before switching on the device. When the device is switchedon, it boots, detects that the durable modules are engaged properly,performs initial diagnostics, and goes into a standby mode. The userthen presses a button near the display to initialize device set-up. TheClinical (OR) Kit enters a mode to allow installation of thedisposables. The user is prompted to scan each disposable componentusing a bar code scanner mounted on the cell separation durable. Whenthe user scans the disposable, the Clinical (OR) Kit will verify thatthe correct durables are in place, then guides the user through eachstep to load the disposable and make necessary tubing connections. Thedevice will sense that the disposable components are properly loaded andensure that all required disposables are installed for the currentapplication. In a preferred embodiment, the barcode scanner is locatedon device such that scanning of the disposables does not interfere withloading of the disposables.

In one embodiment of the cell collection process of the presentinvention, the cell suspension is pumped from the separation module to asyringe in the collection module. A linear actuator pulls the syringeplunger, drawing cell suspension into the syringe. FIG. 5 illustratesthe system flow path.

In the flow path in FIG. 5, which begins after completing device set-up,the user interacts with the user interface to proceed. The deviceperforms an air purge operation in which media and serum are pumpedthrough the flow paths illustrated by the arrows, pushing air to a wastecollection point 508 which has a vent port that allows air to escape tothe atmosphere. The graft chamber 514 is bypassed so that the graft isnever exposed to air.

The user is then prompted to inject adipose 502 into a port on thecentrifuge disposable 506. The adipose tissue 502 is macerated as itenters the centrifuge 506 by passing through stationary blades. In apreferred embodiment, the protease solution is a collagenase/PBSsolution 504. The user interface display indicates that the cellseparation process is initiated.

In a preferred embodiment, from this point on, no user interaction isrequired until the entire Clinical (OR) Kit process is complete. Theuser interface display provides continuous updates on the process,indicating the specific operation being performed, the estimated time tocomplete the operation, and the estimated time to complete the entireprocess. In one embodiment, other important process parameters(temperatures, pump speed, etc.) can also be made available to the uservia the display.

In an embodiment, the graft scaffold is packed in alcohol or otherappropriate sterile substance within the disposable graft chamber 514.Graft preparation is concurrent with the cell separation steps providedbelow. The following steps are involved in preparing the graft forsodding. (1) Alcohol Purge—alcohol is purged from the graft chamber 514by flowing media through the graft chamber 514 and directing the liquidoutlet 520 to waste 508; (2) Scaffold pretreatment media 510, 512 isrecirculated through the graft chamber 514 until the cell suspension isavailable for graft sodding. The media can include, without limitation,M199 (illustrated in FIG. 5), M199E, PBS, Saline, or Di-Cation FreeDPBS. In a preferred embodiment, the media is a 6:1 mixture of MI99E andserum (illustrated in FIG. 5) from the patient.

The cell separation process is identical for sodding and cell collectionoperation modes. In one embodiment, the cell separation steps include:(1) adipose tissue 502 digestion—the centrifuge 506 is temperaturecontrolled at about 37° C. and provides a low speed mixing action(mixing is maintained for an appropriate amount of time to ensureadequate digestion); (2) centrifugation—the centrifuge 506 spins at highRPM, separating the adipose tissue 502 into its constituent materials;and (3) endothelial cell isolation and resuspension. In one embodiment,the separated contents may be directed into a thin, transparent tubewhere an optical sensor detects the location and volume of theendothelial cells. Unwanted materials are directed to a waste 508reservoir, and a specific volume of endothelial cells is returned to thecentrifuge. A 6:1 mixture of MI99E 510: serum 512 is pumped into thecentrifuge. The centrifuge 506 suspends the separated cells in themixture by a low speed mixing action. The cell suspension is then pumpedfrom the centrifuge 506 through a 30-micron filter and directed to thegraft sodding unit or the cell collection unit for collection into asyringe. FIG. 9B shows a cross-sectional view of one embodiment of thecentrifuge bowl.

In the process of graft sodding, liquid passes between the separationmodule and graft module via the sodding module. Preferably, the graft istemperature controlled to about 37° C. In an embodiment, the graftsodding steps include cell sodding and “feed and bleed” flow. In thecell sodding step, the endothelial suspension is introduced into therecirculating flow path, allowing the cell suspension to flow into thegraft at one end and out through the graft walls. Initially, the liquidmixture that leaves the graft chamber is directed to waste until theentire volume of cell suspension has entered the recirculating path. Thecell suspension then recirculates until graft sodding is complete. Themicroporous ePTFE permits the passage of the media/serum mixture, butthe cells are embedded into the ePTFE. During this process, transmuralpressure is monitored by a pressure sensor in the sodding module. In the“feed and bleed” flow step, graft flow is switched to luminal when aspecific transmural pressure is reached, indicating complete sodding.During this process, flow is alternately directed to waste and thepump(s) for recirculation. During periods when the flow is directed towaste, makeup media and serum are pumped from the reservoirs. The “feedand bleed” process is maintained for an appropriate amount of time.

Sustained pressure head, applied to a liquid medium with suspended cellsacross a permeable scaffold material, offers the advantage of rapid celladhesion, without large pressure gradients as used in transient pressuresodding techniques. One skilled in the art could readily practice theinvention with a myriad of cell types, scaffold materials and geometrieswith any number of device designs. Those skilled in the art willrecognize, or be able to ascertain, many equivalents to the specificembodiments of the invention described herein using no more than routineexperimentation. Such equivalents are intended to be encompassed by theclaims.

The present invention provides devices and methods of preparing varioustissue implants or grafts by applying pressure, preferably sustained lowmagnitude pressure, for adhering or “sodding” cells onto any suitablegraft scaffolds or other permeable substrate materials. In a specificembodiment, the tissue is a tubular tissue, such as a vascular tissue.However, the invention is also applicable to any type of tissue graftsinvolving the adhesion of cells to scaffolds or other substratematerials, including, but not limited to, skin, cartilage, bone, bonemarrow, tendon, ligament, gastrointestinal tract, genitourinary tracts,liver, pancreas, kidney, adrenal gland, mucosal epithelium, and nervegrafts. The method is particularly well suited to tubular tissues,including, but not limited to, those of the cardiovascular system andthe urinary system.

The term “sustained low magnitude pressure” as used herein meanspressure having a head of about 10 mmHg, about 15 mmHg, about 20 mmHg,about 25 mmHg and about 30 mmHg and about 55 mmHg, for about 5 min,about 20 min, about 30 min, about 40 min, about 50 min, about I hour,about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 4hours, about 5 hours or about 6 hours, to enhance the adhesion, growthand/or differentiation of the cells. One of ordinary skill in the artcan select appropriate conditions for applying specific low magnitudesustained pressures according to the types of cells, tissue grafts,substrate materials, and given the teachings herein.

The term “transmural pressure or flow” as used herein refers to pressureor flow from one side to the other side of a graft scaffold, across thewall of the graft scaffold. Where the graft scaffold is a tubular graftscaffold, the term refers to pressure or flow from the lumen orintracapillary (IC) space of the graft to the outside or extracapillary(EC) space of the graft.

The term “trans lumenal pressure or flow” as used herein refers topressure or flow through the lumen of a tubular graft. The terms“translumenal flow” and “translumenal perfusion” may be usedinterchangeably. While translumenal perfusion is not required forcellular adhesion in the present invention, it may be used, for example,after the transmural flow to provide a training or cleansing effect. Inthis case, flow rates up to and including physiologic flow rates (−160ml/min) are preferred, although flow rates as low as 5 ml/min typicallyare sufficient to provide cellular adhesion capable of withstandingsubsequent physiologic flow.

Therapeutic Uses

The tissue grafts and cell suspensions prepared by the above-describeddevices can be employed in a myriad of therapeutic uses. For example, inone embodiment of the invention methods are provided for revascularizinga tissue or organ of a subject in need thereof, by implanting into thetissue or organ at least one tissue graft or cell suspension that isprepared by any of the above-described devices. The terms“revascularize”, “revascularizing”, “neovascularization”, or“revascularization” as used herein refer to revising an existingvascular network or establishing a new functional or substantiallyfunctional vascular network in a tissue or organ that has an avascularor hypovascular zone, typically due to disease, congenital defect, orinjury.

In an embodiment, the tissue graft or cell suspension comprises cellsselected from the group consisting of skin, skeletal muscle, cardiacmuscle, atrial appendage of the heart, lung, mesentery, or adiposetissue. The adipose tissue may be from omental fat, properitoneal fat,perirenal fat, pericardial fat, subcutaneous fat, breast fat, orepididymal fat.

In certain embodiments, the tissue graft or cell suspension furthercomprises appropriate stromal cells, stem cells, Relevant Cells, orcombinations thereof. As used herein, the term “stem cells” is used in abroad sense and includes traditional stem cells, adipose derived stemcells, progenitor cells, preprogenitor cells, reserve cells, and thelike. Exemplary stem cells include embryonic stem cells, adult stemcells, pluripotent stem cells, neural stem cells, liver stem cells,muscle stem cells, muscle precursor stem cells, endothelial progenitorcells, bone marrow stem cells, chondrogenic stem cells, lymphoid stemcells, mesenchymal stem cells, hematopoietic stem cells, central nervoussystem stem cells, peripheral nervous system stem cells, and the like.Descriptions of stem cells, including method for isolating and culturingthem, may be found in, among other places, Embryonic Stem Cells, Methodsand Protocols, Turksen, ed., Humana Press, 2002; Weisman et al., Annu.Rev. Cell. Dev. Biol. 17:387403; Pittinger et al., Science, 284:14347,1999; Animal Cell Culture, Masters, ed., Oxford University Press,2000; Jackson et al., PNAS 96 (Shepherd BR et al. Rapid perfusion andnetwork remodeling in a microvascular construct after implantation.Arterioscler. Thromb. Vase Biol. 24: 898-904, 2004): 14482 86, 1999; Zuket al., Tissue Engineering, 7:211 228, 2001 (“Zuk et al.”); Atala andLanza, eds., Academic Press, 2001 (Atala, et al.), particularly Chapters33 41; and U.S. Pat. Nos. 5,559,022, 5,672,346 and 5,827,735.Descriptions of stromal cells, including methods for isolating them, maybe found in, among other places, Prockop, Science, 276:7174, 1997;Theise et al., Hepatology, 31 :235 40, 2000; Current Protocols in CellBiology, Bonifacino et al., eds., John Wiley & Sons, 2000 (includingupdates through March, 2002); and U.S. Pat. No. 4,963,489. The skilledartisan will understand that the stem cells and/or stromal cellsselected for inclusion in a tissue graft or cell suspension aretypically appropriate for the intended use of that construct. In certainembodiments, the tissue graft or cell suspension once implanted in vivo,will develop a functional vascular bed and inosculate with thesurrounding functional vascular system and perfuse, or be capable ofperfusing, the damaged tissue or organ.

According to certain methods for revascularizing tissues or organs, atleast one tissue graft or cell suspension is combined with said tissueor organ and a revascularized tissue or organ is generated. According tocertain methods for revascularizing tissues or organs, the term“combining” comprises placing or implanting at least one tissue graft orcell suspension on any surface of, within, between the layers of, oradjacent to, said tissue or organ. In certain embodiment, the tissuegraft or cell suspension is implanted in the tissue or organ byinjection. In certain embodiments, such injected construct willpolymerize in situ, following implantation. In certain embodiments, suchinjected tissue graft or cell suspension comprises at least one culturedmicrovessel construct, at least one freshly isolated microvesselconstruct, or both. In certain embodiments, combining comprisesattaching at least one tissue graft or cell suspension to at least onetissue or organ in need of revascularizing, using techniques known inthe art, such as described above.

The skilled artisan understands that certain tissues and organs arecovered by or contain a layer of fibrous tissue, connective tissue,fatty tissue, or the like, and that the underlying tissue or organ canbe revascularized without removing this layer. Such a layer may benaturally occurring (such as a serosal layer, mucous membrane, fibrouscapsule, or the like), may result form fibrosis, necrosis, or ischemia,due to disease, defect, injury, or biochemical deficiency. Typically,the microvessel fragments of the tissue graft or cell suspension canpenetrate such a layer and inosculate with the vasculature of theunderlying tissue or organ, revascularizing the tissue or organ. Thus,combining the tissue graft or cell suspension with the tissue or organin need of revascularization, comprises placing the tissue graft or cellsuspension on or in such layer. For example, but not limited to, placingthe tissue graft or cell suspension on the meninges to revascularizebrain tissue; the epicardium to revascularize the myocardium; theperitoneum and/or serosa, to revascularize portions of the largeintestine; the conjunctiva and/or subconjunctiva to revascularize theeye; the tracheal surface to revascularize the trachea; the bucchalmucosa to revascularize the mouth; the pleural and/or serosal surface torevascularize the lung; the pleural and/or peritoneal surface torevascularize the diaphragm; the skin to revascularize non-healing skinulcers, such as diabetic ulcers; the pericardial surface torevascularize the pericardium; and the like.

In certain embodiments, the tissue graft or cell suspension, whencombined with the tissue or organ within the animal or human, willdevelop functional vascular bed and inosculate with the surroundingfunctional vascular system and perfuse the damaged tissue or organ. Incertain embodiments, the implanted tissue graft or cell suspensionserves as a nucleation site for revascularizing the damaged tissue ororgan. In certain embodiments, appropriate stem cells, stromal cells,and/or Relevant Cells from the tissue graft or cell suspension willsupport the restructuring and repair of the damaged tissue or organ.Constructs comprising genetically engineered cells may producerecombinant products that are distributed systemically via thebloodstream or delivered to the local microenvironment to induce repair,wound healing, or the like.

In a particular embodiment, the tissue graft or cell suspensioncomprises endothelial cells which are capable of differentiating into,without limitation, a neuron, myocardiocyte, chondrocyte, pancreaticancinar cell, pancreatic endocrine cells including islet of Langer hans,hepatocyte, renal epithelial cell, parathyroid cell, Leydig cell,sertoli cell, gonocyte, oocyte, blastocyst, Kupffer cell, lymphocyte,fibroblast, myocyte, myoblast, satellite cell, adipocyte, preadipocyte,osteocyte, osteoblast, osteoclast, chondrocyte, biliary epithelial cell,Purkinje cell, and pacemaker cell

In another particular embodiment, the tissue graft or cell suspensioncomprises at least one stem cell, progenitor cell or Relevant Cell,which may be without limitation a neuron, myocardiocyte, chondrocyte,pancreatic ancinar cell, pancreatic endocrine cells including islet ofLangerhans, hepatocyte, renal epithelial cell, parathyroid cell, Leydigcell, sertoli cell, gonocyte, oocyte, blastocyst, Kupffer cell,lymphocyte, fibroblast, myocyte, myoblast, satellite cell, adipocyte,preadipocyte, osteocyte, osteoblast, osteoclast, chondrocyte, biliaryepithelial cell, Purkinje cell, and pacemaker cell.

The term “Relevant Cell(s)” as used herein refers to cells that areappropriate for incorporation into a tissue graft or cell suspensionprepared by the devices of the present invention, based on the intendeduse of that tissue graft or cell suspension. By way of example, RelevantCells that are appropriate for the repair, restructuring, orrepopulation of damaged liver may include, without limitation,hepatocytes, biliary epithelial cells, Kupffer cells, fibroblasts, andthe like. Exemplary Relevant Cells for incorporation into tissue graftor cell suspensions include neurons, myocardiocytes, myocytes,chondrocytes, pancreatic acinar cells, islets of Langerhans, osteocytes,hepatocytes, Kupffer cells, fibroblasts, myocytes, myoblasts, satellitecells, endothelial cells, adipocytes, preadipocytes, biliary epithelialcells, and the like. These types of cells may be isolated and culturedby conventional techniques known in the art. Exemplary techniques can befound in, among other places, Atala et al., particularly Chapters 9-32;Freshney, Culture of Animal Cells A Manual of Basic Techniques, 4th ed.,Wiley Liss, John Wiley & Sons, 2000; Basic Cell Culture: A PracticalApproach, Davis, ed., Oxford University Press, 2002; Animal CellCulture: A Practical Approach, Masters, ed., 2000; and U.S. Pat. Nos.5,516,681 and 5,559,022.

The skilled artisan will appreciate that such stromal cells, stem cells,and/or Relevant Cells may be incorporated into the tissue graft or cellsuspension during or after preparation. For example, but not limited to,combining the cell suspension, stem cells, Relevant Cells, and/orstromal cells in a liquid three-dimensional culture, such as coHagen,fibrin, or the like, or seeding or sodding stem cells, Relevant Cells,and/or stromal cells in or on the tissue graft may be achieved.Exemplary combinations of appropriate stem cells, stromal cells, andRelevant Cells for incorporation into tissue grafts or cell suspensionsinclude: islets of Langerhans and/or pancreatic acinar cells in a tissuegraft or cell suspension for revascularizing a damaged pancreas;hepatocytes, hepatic progenitor cells, Kupffer cells, endothelial cells,endodermal stem cells, liver fibroblasts, and/or liver reserve cells ina tissue graft or cell suspension for revascularizing a damaged liver.For example, but not limited to, appropriate stem cells or stromal cellsfor a tissue graft or cell suspension for vascularizing, repairing, andreconstructing a damaged or disease liver might comprise liver reservecells, liver progenitor cells, such as, but not limited to, liverfibroblasts, embryonic stem cells, liver stem cells, cardiomyocytes,Purkinje cells, pacemaker cells, myoblasts, mesenchymal stem cells,satellite cells, and/or bone marrow stem cells for revascularizing adamaged or ischemic heart (see, e.g., Atkins et al., J. of Heart andLung Transplantation, December 1999, at pages 1173 80; Tomita et al.,Cardiovascular Research Institute, American Heart Association, 1999, atpages 92 101; Sakai et al., Cardiovascular Research Institute, AmericanHeart Association, 1999, at pages 108 14), and the like.

In one embodiment, the tissue graft or cell suspension further comprisesan agent selected from the group consisting of cytokines, chemokines,antibiotics, drugs, analgesic agents, anti-inflammatory agents,immunosuppressive agents, or combinations thereof. Exemplary cytokinesmay include, without limitation, angiogenin, vascular endothelial growthfactor (VEGF, including, but not limited to VEGF-165), interleukins,fibroblast growth factors, for example, but not limited to, FGF-1 andFGF-2, hepatocyte growth factor, (HGF), transforming growth factor beta(TGF-.beta.), endothelins (such as ET-1, ET-2, and ET-3), insulin-likegrowth factor (IGF-1), angiopoietins (such as Ang-1, Ang-2, Ang-3/4),angiopoietin-like proteins (such as ANGPTL1, ANGPTL-2, ANGPTL-3, andANGPTL-4), platelet-derived growth factor (PDGF), including, but notlimited to, PDGF-AA, PDGF-BB and PDGF-AB, epidermal growth factor (EGF),endothelial cell growth factor (ECGF), including ECGS, platelet-derivedendothelial cell growth factor (PD-ECGF), placenta growth factor (PLGF),and the like. Cytokines, including recombinant cytokines, and chemokinesare typically commercially available from numerous sources, for example,R&D Systems (Minneapolis, Minn.); Endogen (Woburn, Wash.); and Sigma(St. Louis, Mo.). The skilled artisan will understand that the choice ofchemokines and cytokines for incorporation into particular tissue graftor cell suspensions will depend, in part, on the target tissue or organto be vascularized, revascularized, augmented or reconstructed.

In certain embodiments, tissue graft or cell suspensions furthercomprise at least one genetically engineered cell. In certainembodiments, tissue graft or cell suspensions comprising at least onegenetically engineered cell will constitutively express or induciblyexpress at least one gene product encoded by at least one geneticallyengineered cell due to the genetic alterations within at least onegenetically engineered cell induced by techniques known in the art.Descriptions of exemplary genetic engineering techniques can be foundin, among other places, Ausubel et al., Current Protocols in MolecularBiology (including supplements through March 2002), John Wiley & Sons,New York, N.Y., 1989; Sambrook et al., Molecular Cloning: A LaboratoryManual, 2.sup.nd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989; Sambrook and Russell, Molecular Cloning: ALaboratory Manual, 3.sup.rd Ed., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 2001; Beaucage et al., Current Protocols inNucleic Acid Chemistry, John Wiley & Sons, New York, N.Y., 2000(including supplements through March 2002); Short Protocols in MolecularBiology, 4.sup.th Ed., Ausbel, Brent, and Moore, eds., John Wiley &Sons, New York, N.Y., 1999; Davis et al., Basic Methods in MolecularBiology, McGraw Hill Professional Publishing, 1995; Molecular BiologyProtocols (see the highveld.com website), and Protocol Online(protocol-online.net). Exemplary gene products for genetically modityingthe genetically engineered cells of the invention include plasminogenactivator, soluble CD4, Factor VIII, Factor IX, von Willebrand Factor,urokinase, hirudin, interferons, including alpha-, beta- andgamma-interferon, tumor necrosis factor, interleukins, hematopoieticgrowth factor, antibodies, glucocerebrosidase, adenosine deaminase,phenylalanine hydroxylase, human growth hormone, insulin,erythropoietin, VEGF, angiopoietin, hepatocyte growth factor, PLGF, andthe like.

In an embodiment of the invention, the tissue or organ is selected fromthe group consisting of heart tissue, lung tissue, cardiac muscletissue, striated muscle tissue, liver tissue, pancreatic tissue,cartilage, bone, pericardium, peritoneum, kidney, smooth muscle, skin,mucosal tissue, small intestine, and large intestine and adipose tissue.

The step of injecting a cell suspension into a subject tissue or organmay include, without limitation, using at least one syringe, needle,cannula, catheter, tube, or microneedle. The terms “injecting”,“injection”, or variations thereof as used herein shall refer to anymeans of ejecting or extruding a substance, typically through a tube orstructure comprising a bore or external opening. Such tube or structurecan be flexible, inflexible, or can comprise at least one flexibleportion and at least one inflexible portion. Exemplary injection meansinclude a syringe with or without a needle, a cannula, a catheter,flexible tubing, and the like. Delivery of the particular cellsuspension might also be accomplished through the use of devices thatpermeablize tissue, such as microneedles. In contrast to traditionalinjections with standardgauge hypodermic needles, microneedle (typicallydefined by a radius of curvature about 1 μm) or microneedle arrayspermeabilize the skin or endothelial cell layer by producing microscopicholes. These holes, in effect, act as conduits for materials deliveryand may enhance the attachment or delivery of a cell suspension of thepresent invention to a vessel, tissue, or organ. Thus, the skilledartisan will understand that any structure comprising a bore or externalopening through which at least one cell suspension can be extruded on orinto a tissue or organ, or any structure that can permeabilize thesurface of a tissue or and organ, including an engineered tissue, iswithin the intended scope of the invention. In certain embodiments, suchinjected construct polymerizes in vitro, following injection.

In a particular embodiment, the tissue graft or cell suspension of thepresent invention comprises cells selected from the group consisting ofskin, skeletal muscle, cardiac muscle, atrial appendage of the heart,lung, mesentery, or adipose tissue. The adipose tissue may be selectedfrom the group consisting of omental fat, properitoneal fat, perirenalfat, pericardial fat, subcutaneous fat, breast fat, or epididymal fat.

Also provided are methods for augmenting a tissue or organ of a subjectin need thereof, comprising implanting into the organ or tissue a tissuegraft prepared by the devices of the present invention or injecting intothe tissue or organ a cell suspension prepared by the devices of thepresent invention. As used herein, “augmenting” refers to increasing thevolume and/or density of the tissue or organ.

Methods are also provided for regenerating a tissue or organ in asubject by implanting into the tissue or organ at least one tissue graftprepared by the devices described herein or by injecting into the tissueor organ at least one cell suspension prepared by the devices of theinvention. As used herein, “regenerating” refers to replacing lost,diseased or otherwise damaged tissue by the formation of new tissue.

A skilled artisan will appreciate that the subject of the presentinvention may be any animal, including amphibians, birds, fish, mammals,and marsupials, but is preferably a mammal (e.g., a human; a domesticanimal, such as a cat, dog, monkey, mouse, and rat; or a commercialanimal, such as a cow, horse or pig). Additionally, the subject of thepresent invention may be of any age, including a fetus, an embryo, achild, and an adult. In a preferred embodiment of the present invention,the subject is human. In one embodiment, the subject is a horse andmethods of the subject invention are used to regenerate tissues in andaround the hooves of the animal. In further embodiments, the subject isa human, and the methods of tissue regeneration are used to prevent ortreat, for example arthritis and diseases of the eye, including but notlimited to, glaucoma and macular degeneration.

Additionally, methods for reconstructing a tissue or organ in a subjectin need thereof comprising implanting into the tissue or organ at leastone tissue graft prepared by the devices described herein or byinjecting into the tissue or organ at least one cell suspension preparedby these devices. As used herein, “reconstructing” refers to rebuilding,reconstituting, reshaping and/or restoring a tissue or organ. In oneembodiment of the invention, for example the subject has cellulite, andthe subject is administered a subcutaneuous injection of an appropriatecell suspension in order to locally reconstruct the adipose tissue, thusimproving the cosmetic appearance of the subject. In one embodiment, thesubject is a post-surgical subject.

Also provided are methods for treating or preventing primary andsecondary infections in a tissue or organ of a subject by implantinginto the tissue or organ at least one tissue graft prepared by thedevices described herein or by injecting into the tissue or organ atleast one cell suspension prepared by these devices.

Methods for using the cell suspensions and tissue grafts prepared by thedevices of the present invention to prevent the formation of scar tissuein a tissue or organ, and/or to treat or prevent inflammation in atissue or organ of a subject are also provided.

Also provided are methods for preventing adhesion formation in a tissueor organ of a subject in need thereof by injecting into the tissue ororgan at least one cell suspension or tissue graft prepared by thedevices of the invention.

In one embodiment, a method is provided for treating or preventing acutemyocardial infarction in a subject by injecting into the heart at leastone cell suspension prepared by any of the devices described herein,wherein vasculature to the heart tissue is increased. In anotherembodiment, methods for treating myocarditis in a subject are providedcomprising injecting into the pericardial fluid of the subject at leastone cell suspension prepared by any of the devices of present invention.

Methods for treating a wound in a subject by injecting the wound with atleast one cell suspension prepared by the device of the presentinvention are also provided. In one embodiment, the subject is apost-surgical subject.

The current invention provides sustained pressure sodding and automationof the clinical procedures of separating a desired fraction of thepatient's cells from tissue and filtering, rinsing, heating, macerating,proteolytically releasing, separating, resuspending, and pressuresodding the cells onto a permeable graft. Those skilled in the art willrecognize, or be able to ascertain, many equivalents to the embodimentsof the inventions described herein using no more than routineexperimentation. Such equivalents are intended to be encompassed by thefollowing claims. All publications, patents and patent applicationsmentioned in this specification are herein incorporated by referenceinto the specification to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference.

The above description and example are only illustrative of preferredembodiments which achieve the objects, features, and advantages of thepresent invention, and it is not intended that the present invention belimited thereto.

What is claimed is: 1.-4. (canceled)
 5. A method comprising: agitating acentrifuge comprising adipose tissue and collagenase for a predeterminedtime period to allow for the digestion of the adipose tissue by thecollagenase to form a digested material; removing the digested materialfrom the centrifuge bowl; filtering the removed digested material byremoving a plurality of fibrous tissue via a first filter; disposing,subsequent to filtering, the digested material in the centrifuge bowl;separating a plurality of cells from the digested material in thecentrifuge bowl; collecting the plurality of cells.
 6. The method ofclaim 5, further comprising separating the plurality of cells from thedigested material, wherein the plurality of cells comprises fibroblasts,smooth muscle cells, pericytes, macrophages, monocytes, plasma cells,mast cells, adipocytes, tissue-specific parenchymal cells, endothelialcells, urothelial cells, or adipose derived stem cells
 7. The method ofclaim 6, wherein the stem cells comprise: embryonic stem cells, adultstem cells, pluripotent stem cells, neural stem cells, liver stem cells,muscle stem cells, muscle precursor stem cells, endothelial progenitorcells, bone marrow stem cells, chondrogenic stem cells, lymphoid stemcells, mesenchymal stem cells, hematopoietic stem cells, central nervoussystem stem cells, peripheral nervous system stem cells
 8. The method ofclaim 5, further comprising: disposing a first mixture into thecentrifuge to create a suspension including the plurality of cells andfiltering the suspension via a second filter.
 9. The method of claim 8,further comprising forming a graft from the plurality of collectedcells, wherein forming the graft comprises: pressure sodding a graftsubstrate by applying sustained low magnitude pressure to the suspensionas it flows across the graft substrate to form a graft; circulating asecond mixture over the pressure sodded graft substrate; and harvestingthe graft, wherein the second mixture is circulated over the pressuresodded graft until the graft is harvested.
 10. The method of claim 9,further comprising, prior to pressure sodding the substrate, pretreatingit with at least one of M199, M199E, PBS, Saline, or Di-Cation FreeDPBS.
 11. The method of claim 9, further comprising pressure sodding thegraft substrate, wherein the graft substrate comprises skin, cartilage,bone, bone marrow, tendon, ligament, gastrointestinal tract,genitourinary tracts, liver, pancreas, kidney, adrenal gland, mucosalepithelium, or nerve grafts.
 12. The method of claim 9, furthercomprising pressure sodding the graft substrate with at least 200,000cells.
 13. A method of preparing a graft, comprising: agitating acentrifuge comprising adipose tissue and collagenase for a predeterminedtime period to allow for the digestion of the adipose tissue by thecollagenase to form a digested material; removing the digested materialfrom the centrifuge bowl; filtering the digested material by removing aplurality of fibrous tissue via a first filter; disposing, subsequent tofiltering, the digested material back in the centrifuge bowl; separatinga plurality of endothelial cells from the digested material in thecentrifuge bowl; disposing a first mixture into the centrifuge to createa suspension including the plurality of endothelial cells; filtering thesuspension via a second filter; pressure sodding a graft by applyingsustained low magnitude pressure to a graft scaffold in a graft chamberto form a graft; circulating a second mixture over the pressure soddedgraft scaffold; and harvesting the graft, wherein the second mixture iscirculated over the pressure sodded graft until the graft is harvested.14. The method of claim 5, further comprising disposing a portion of thedigested material in a pristine waste bag subsequent to filtering. 15.The method of claim 14, further comprising disposing the digestedmaterial from the pristine waste bag in the centrifuge bowl.
 16. Themethod of claim 14, further comprising disposing the first mixturecomprising M199 and serum into the centrifuge.
 17. The method of claim14, further comprising disposing the first mixture comprising M199 andserum into the centrifuge in a ration of M199:serum of 6:1.
 18. Themethod of claim 14, further comprising disposing the first mixturecomprising serum and at least one of M199, M199E, PBS, Saline, orDi-Cation Free DPBS.
 19. The method of claim 14, further comprisingdisposing, subsequent to separating, the plurality of endothelial cellsin a tube and determining, via an optical sensor, a location and avolume of the plurality of endothelial cells.
 20. The method of claim17, further comprising disposing the plurality of endothelial cells intothe centrifuge subsequent to determining the location and the volume.21. The method of claim 13, further comprising filtering the removeddigested material to remove the plurality of fibrous tissue greater than100 μm.
 22. The method of claim 13, further comprising filtering thesuspension via the second filter to remove particles greater than 30 μm.23. The method of claim 13, further comprising, subsequent to pressuresodding the graft scaffold, purging a plurality of epithelial cells. 24.The method of claim 13, further comprising circulating the secondmixture comprising M199 and serum over the pressure sodded graftscaffold.